LOCUS       sequence-118373-        3742 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses gRNA in Leishmania.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3742)
  AUTHORS   Zhang WW, Matlashewski G
  TITLE     CRISPR-Cas9-Mediated Genome Editing in Leishmania donovani.
  JOURNAL   MBio. 2015 Jul 21;6(4). pii: e00861-15. doi: 10.1128/mBio.00861-15.
  PUBMED    26199327
REFERENCE   2  (bases 1 to 3742)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "MBio."; 
            date: "2015-07-21"; volume: "6(4). pii: e00861-15. doi"; pages: " 
            10.1128/mBio.00861-15"
COMMENT     Sequence Label: pSPneoHHgRNAH
FEATURES             Location/Qualifiers
     source          1..3742
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        2..20
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     2..19
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     ncRNA           241..277
                     /product="self-cleaving hammerhead ribozyme (Ferre-D'Amare 
                     and Scott, 2010)"
                     /label=hammerhead ribozyme
                     /ncRNA_class="hammerhead_ribozyme"
     misc_RNA        302..377
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     ncRNA           384..451
                     /product="hepatitis delta virus self-cleaving ribozyme 
                     (Ferre-D'Amare and Scott, 2010)"
                     /label=HDV ribozyme
                     /ncRNA_class="ribozyme"
     CDS             573..1367
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(627..646)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     1237..1256
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(1397..1416)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1398..1416)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1503..1520)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1674..2262)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1754..1773)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2433..3224,3225..3293))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     3056..3075
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3294..3398)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3466..3484
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(3522..3544)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     3644..3663
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gatttaggtg acactataga actcgagcag ctgaagcttg tgagttatga ggtctgcgat
       61 tgacgtagga gttgcaaggg ggagggggta tgaacggggt gggtagagct tttttaggtg
      121 gaagtagtga gagggtgggc ttgtgagaat ttgaggtgtg ttcgtgatgt gtggatctta
      181 tcgggggctc ggttgagttt ttggtttggt gattttgtag gaagcttaag atctcaccat
      241 ctgatgagtc cgtgaggacg aaacgagtaa gctcgtcatg gtggggtctt cgagaagacc
      301 tgttttagag ctagaaatag caagttaaaa taaggctagt ccgttatcaa cttgaaaaag
      361 tggcaccgag tcggtgcttt tttggccggc atggtcccag cctcctcgct ggcgccggct
      421 gggcaacatg cttcggcatg gcgaatggga cggatccccg ggtaccgagc tctctcttct
      481 ccttcccctc tctctcctct ctcctctctc cagagctctc tcttctcctt cccctctctc
      541 tcctctctcc tctctccaga gctcgaattc gcatgattga acaagatgga ttgcacgcag
      601 gttctccggc cgcttgggtg gagaggctat tcggctatga ctgggcacaa cagacaatcg
      661 gctgctctga tgccgccgtg ttccggctgt cagcgcaggg gcgcccggtt ctttttgtca
      721 agaccgacct gtccggtgcc ctgaatgaac tgcaggacga ggcagcgcgg ctatcgtggc
      781 tggccacgac gggcgttcct tgcgcagctg tgctcgacgt tgtcactgaa gcgggaaggg
      841 actggctgct attgggcgaa gtgccggggc aggatctcct gtcatctcac cttgctcctg
      901 ccgagaaagt atccatcatg gctgatgcaa tgcggcggct gcatacgctt gatccggcta
      961 cctgcccatt cgaccaccaa gcgaaacatc gcatcgagcg agcacgtact cggatggaag
     1021 ccggtcttgt cgatcaggat gatctggacg aagagcatca ggggctcgcg ccagccgaac
     1081 tgttcgccag gctcaaggcg cgcatgcccg acggcgagga tctcgtcgtg acccatggcg
     1141 atgcctgctt gccgaatatc atggtggaaa atggccgctt ttctggattc atcgactgtg
     1201 gccggctggg tgtggcggac cgctatcagg acatagcgtt ggctacccgt gatattgctg
     1261 aagagcttgg cggcgaatgg gctgaccgct tcctcgtgct ttacggtatc gccgctcccg
     1321 attcgcagcg catcgccttc tatcgccttc ttgacgagtt cttctgatcg atgatatcag
     1381 atcgatctgc cggtctccct atagtgagtc gtattaattt cgataagcca ggttaacctg
     1441 cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct
     1501 tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac
     1561 tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga
     1621 gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat
     1681 aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac
     1741 ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct
     1801 gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg
     1861 ctttctcaat gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg
     1921 ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt
     1981 cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg
     2041 attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac
     2101 ggctacacta gaaggacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga
     2161 aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt
     2221 gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt
     2281 tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga
     2341 ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc
     2401 taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct
     2461 atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata
     2521 actacgatac gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca
     2581 cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga
     2641 agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga
     2701 gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg
     2761 gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga
     2821 gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt
     2881 gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct
     2941 cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca
     3001 ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat
     3061 accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga
     3121 aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc
     3181 aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg
     3241 caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc
     3301 ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt
     3361 gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca
     3421 cctgacgtct aagaaaccat tattatcatg acattaacct ataaaaatag gcgtatcacg
     3481 aggccctttc gtctcgcgcg tttcggtgat gacggtgaaa acctctgaca catgcagctc
     3541 ccggagacgg tcacagcttg tctgtaagcg gatgccggga gcagacaagc ccgtcagggc
     3601 gcgtcagcgg gtgttggcgg gtgtcggggc tggcttaact atgcggcatc agagcagatt
     3661 gtactgagag tgcaccatat ggacatattg tcgttagaac gcggctacaa ttaatacata
     3721 accttatgta tcatacacat ac
//
